/ . EmbryoI. exp. Morph. Vol. 50, pp. 169-173, 1979
Printed in Great Britain © Company of Biologists Limited 1979
Neurotrophic and X-ray blocks in
the blastemal cell cycle
By M. MADEN 1
From The National Institute for Medical Research, London
SUMMARY
Using microdensitometry techniques the points in the cycle where blastemal cells become
blocked after X-irradiation or denervation of the regenerating amphibian limb have been
identified. X-irradiation blocks the cells in both Gx and G2 and those cells that were in S at
the time of irradiation presumably proceed to G2. After denervation, however, cells accumulate
only in Gx and those that were in S or G2 continue through the cycle to the next Gj.. The
latter results are clearly contradictory to a recent theory proposing a G2 neurotrophic control
of blastemal cells and a solution to the contradiction is presented in the light of recent
results.
INTRODUCTION
Regeneration of the amphibian limb is an epimorphic phenomenon and
hence dependent upon a continued supply of new cells for restitution of the
original pattern. DedifTerentiated cells of the stump provide this material by
mitosis and a blastema appears at the tip of the limb. Thus if cell division is
inhibited no regeneration will occur - a situation which prevails when the limb
is either denervated or X-irradiated. Following denervation of the blastema,
DNA synthesis declines (Singer & Caston, 1972) and the mitotic index falls to
zero (Singer & Craven, 1948): a newt brain extract injected into the blastema
reinitiates synthesis (Jabaily & Singer, 1977). Similarly after irradiation with a
suitable dose of X-rays (> 1200R) a mitotic block is incurred (Maden & Wallace
1976; Wertz, Donaldson & Mason, 1976) and it is thus tempting to speculate
that these two inhibitory treatments act in a similar fashion on some key point
in the blastemal cell cycle. However, I report here that whereas irradiation
blocks cells both in the Gx or G2 phases of the cell cycle, denervated blastemal
cells accumulate only in Gv This latter observation is clearly contrary to a
recent theory proposing that nerves control the G2 phase of the blastemal cell
cycle (Tassava & Mescher, 1975).
1
Author's address: Division of Developmental Biology, National Institute for Medical
Research, The Ridgeway, Mill Hill, London, NW7 1AA, U.K.
170
M. MADEN
MATERIALS AND METHODS
The forelimbs of 12 axolotls, Ambystoma mexicanum, were amputated through
the distal humerus and allowed to regenerate at 20 °C until cone stage blastemas
had developed. Five animals then had their forelimbs denervated by crushing
the nerves at the brachial plexus. The other five were irradiated with 2000R
from a Newton and Victor Ltd X-ray machine at 90 kV, 5-5 mA. Two blastemas
in each series were sampled at daily intervals for four days including one sample
immediately after treatment (day 0). The remaining two animals were used as
additional controls fixed on day 4. All blastemas were fixed in 3:1 alcohol: acetic
acid, hydrolysed in 5 N-HC1 for 1 h and stained in Feulgen for 2 h. They were
tapped out in water after removing the epidermis and squashed under a coverslip.
The absorption values of 100 single nuclei on each sample were read on a
Vickers M85 scanning microdensitometer to determine the proportion of cells
in each phase of the cell cycle. The cycle time of young axolotl blastemal cells is
2 days (Maden, 1976), thus by 4 days after treatment all the cells should have
collected at any arrest point.
RESULTS
Figs. \a and 2 a are the histograms from control blastemas (day 0) showing
typical distributions of DNA contents in rapidly dividing populations of cells
(day 4 controls also showed these characteristics). It has previously been
revealed (Maden, 1976) that in these blastemal cells the duration of S phase is
about 60 % of the total cycle time. We would therefore not expect any obvious
bimodality in the Feulgen distributions of normal blastemas.
Irradiation. After irradiation (Figs. 1 b and 1 c) there was a gradual appearance
of two distinct peaks as cells seemed to be lost from the middle (S phase). This
suggests that cells which were in Gx or G2 at the time of irradiation are prevented
from proceeding further through the cycle and cells undergoing DNA synthesis
continue, perhaps to G2, and then stop. However, this interpretation is probably
too simplistic since in Fig. 1 c the proportion of cells in Gx (left-hand peak)
is 65 % and G2 (right-hand peak) 35 %. Had all those cells which were in S phase
at the time of irradiation (60 % of the population - see above) progressed to
G 2 and then stopped there would clearly be far more cells in G2 than Gv
Although the reason for the larger G± peak observed here remains to be elucidated, we can conclude that there seem to be two blocks after blastemal cells
are irradiated.
Denervation. Following denervation, on the other hand, both the S phase and
and the G2 cells gradually disappeared (Fig. 2b) leaving one massive Gx peak
(Fig. 2 c). By 4 days after denervation almost all the cells were in Gx and the
height of this peak had increased threefold. Thus in contrast to the effects of
irradiation described above, after denervation there seems only to be one block
point and that is in Gx.
Nerves, X-rays and the cell cycle
60
111
120
Absorption class
Fig. 1. Irradiation: Frequency of nuclei in classes of Feulgen absorption from.
(a) normal cone blastema, (b) blastema 2 days after irradiation with 2000R,
(c) blastema 4 days after irradiation. (6) and (c) show that irradiated cells become
blocked in Gx and G2.
40-
=
(b)
(a)
(c)
20 •
60
120
Absorption class
ISO
Fig. 2. Denervation: Frequency of nuclei in classes of Feulgen absorption from
(a) normal cone blastema, (b) blastema 2 days after denervation, (c) blastema 4 days
after denervation. (6) and (c) show that cells collect in Gx after denervation.
DISCUSSION
Radiobiological studies in other tissues (Davies & Evans, 1966) have identified
both Gx and G2 blocks after irradiation and the above data show that axolotl
blastemas are typical in this regard. These results complement the demonstration
that after irradiation the amputated limb does not initiate DNA synthesis as
normal (Wertz et ah 1976) and neither do mitoses appear (Maden & Wallace,
1976). The reason for this inhibition is therefore clear - the cells at the tip of
the limb cannot leave the Qx phase (or Go) in which they have differentiated.
Concerning the effects of denervation, it has been concluded by Tassava and
his colleagues (Tassava, Bennett & Zitnik, 1974; Mescher & Tassava, 1975;
172
M. MADEN
Tassava & Mescher, 1975) that a neurotrophic factor is required for the passage
of cells through the G2 phase of the blastemal cell cycle. They arrived at this
conclusion on the basis of the observation that when denervated limbs were
amputated, the cells at the tip began to synthesise DNA but no mitoses appeared.
However, this hypothesis has recently been challenged following the repetition
and extension of this type of experiment (Maden, 1978). Not only were labelled
cells and mitoses observed after denervated limbs were amputated, but denervation 6 days prior to amputation reduced the proportion of labelled cells and
number of mitoses. It was therefore suggested that there is a pool of trophic
factor in the limb. Now the results reported here show that denervated cells
become blocked in Gl5 so if the pool of trophic factor were to be totally removed
from the limb before amputation there should be no cells entering S phase or
mitosis. This experiment has yet to be performed. Thus it seems that this pool
of trophic factor is responsible for the spurious observation of labelled cells in
denervated limbs (Tassava et a/. 1974) which led to the hypothesis of a G2
trophic control.
Mescher & Tassava (1975) themselves report the results of microdensitometric
analysis of sections of denervated limbs. They failed to show cells collecting in
G2 and suggested the reason for this failure was that G2 blocked cells were
selectively removed from the limb. This was not the case in G2 blocked irradiated
cells (Fig. 1 c) which remained in the blastema, implying selective removal is not
a real mechanism. However, microdensitometry of sections is notoriously inaccurate; indeed neither did Mescher & Tassava's data reveal a Gx block. This
problem was overcome here by the use of blastemal squashes to demonstrate
that denervated cells accumulate in G±.
Finally, the question of whether the trophic factor is a rate-limiting (by
controlling the rate of G2) or an all-or-nothing molecule (by controlling the
number of cells cycling) has already been discussed (Maden, 1978). It is
important to emphasize that the data reported here do not distinguish between
these two possibilities as a greatly protracted G± phase would accumulate cells
in G2 just as a complete block. Other evidence from continous labelling studies
does, however, favour the rate-limiting hypothesis (Maden, unpublished).
In conclusion it is clear that although denervation and irradiation both
inhibit cell division and hence limb regeneration, they seem to do so by different
cellular-mechanisms.-The above results must surely lay to rest the hypothesis
(Rose, 1962) that X-rays act by destroying the nervous component of the limb.
I thank Dr H. Wallace for expert guidance in the use of the microdensito meter and Prof.
J. Jinks for the use of facilities in the Department of Genetics, University of Birmingham.
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{Received 4 August 1978, revised 10 October 1978)
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